Diagnostic and therapeutic use of antibodies against the urokinase receptor

ABSTRACT

The invention concerns a method and a reagent kit for detecting cells in a biological sample using a double-fluorescence technique and the diagnostic and therapeutic application of amino acid sequence-specific antibodies against the urokinase receptor having a high affinity for tumour cell-expressed receptors.

[0001] The invention concerns a method and a reagent kit for detectingcells in a biological sample using a double-fluorescence technique.

[0002] The reliable detection of disseminated tumour cells which haveescaped from a solid tissue structure (micrometastases) is of majorimportance for tumour diagnostics and treatment. Hence various methodshave been developed over the past years to detect such individualdisseminated tumour cells in body fluids or tissue samples. They can forexample be detected by selectively labelling the rare cells by means ofimmunocytochemical methods in which case enzymatic labelling groups suchas alkaline phosphatase are often used. Double labelling techniques arealso known.

[0003] A publication by Schlimok et al. (Proc. Natl. Acad. Sci. USA 84(1987) 8672-8676) describes the detection of micrometastatic tumourcells in bone marrow by means of a double-labelling technique in which acytokeratin 18 antibody which is specific for cells of epidermal originand a leucocyte antibody are used. In this method alkaline phosphataseand a radioactive labelling group (¹²⁵I) are used. Since there aredrawbacks associated with the use of radioactive labelling groups, thismethod is not suitable for clinical practice.

[0004] Funke et al. (Int. J. Cancer 65 (1996), 755-761) describe thedetection of micrometastases in bone marrow by means of adouble-labelling technique using a cytokeratin 18 antibody and anE-cadherin antibody. Both antibodies are detected by means of alkalinephosphatase as an enzymatic labelling group and two differently colouredchromogenic substrates. However, the sequential detection of bothantibodies by different chromogenic substrates is complicated and henceless suitable for clinical practice.

[0005] Heiss and co-workers (Heiss et al., Nature Med. 1 (1995),1035-1039 and Allgayer et al., J. Histochem. Cytochem. 45 (1997),203-212) detect disseminated tumour cells in bone marrow by means of adouble-labelling method based on the simultaneous detection ofcytokeratin 18 and the uPA receptor (uPAR). For this cells bound andfixed on a microscope slide are incubated with a biotinylatedcytokeratin-specific antibody and subsequently with a conjugate ofalkaline phosphatase and streptavidin. An enzymatic staining reaction iscarried out using the immobilized alkaline phosphatase and a chromogenicsubstrate to form a dark-red stain. In addition a monoclonal antibodyagainst uPAR is used which is labelled with a gold-conjugated secondaryantibody and subsequently subjected to a silver enhancement reactionwhich results in a black stain. The microscope slides are then manuallyand visually screened for the stains (dark-red/black) under a microscopebut a double stain is extremely difficult to detect.

[0006] The object of the present invention was to carry out a method forthe detection of cells in particular of rarely occurring cells such astumour cells in a biological sample e.g. bone marrow which at leastpartially eliminates the disadvantages of the prior art. In particularthe method should at the same time be highly sensitive and enable atroublefree evaluation.

[0007] This object is achieved by a method for detecting cells in abiological sample which comprises the following steps:

[0008] (a) preparing a sample to be tested,

[0009] (b) contacting the sample with at least two different bindingmolecules which recognize the cells to be detected, the bindingmolecules being each labelled with different fluorescent dyes and

[0010] (c) determining the fluorescent labels in the sample fixed on asolid phase.

[0011] The method according to the invention is suitable for detectingrarely occurring cells in a fixed biological sample. In this connection“rarely occurring” in the sense of the present invention means that theexpected frequency of the cells to be detected is in the range of 1:10⁴to 1:10⁷ of the total number of cells present in the sample to bedetected. Examples of such rarely occurring cells are tumour cells in ablood or bone marrow sample. Other types of rarely occurring cells canof course also be detected if the cell-specific determinants andspecific binding molecules are selected accordingly.

[0012] The double-fluorescent staining technique of the method accordingto the invention allows a rapid and accurate identification of the cellsto be detected. In addition the use of different fluorescent labels thatcan be preferably detected concurrently enables antigens to be analysedthat are co-located in as well as on the cell (such as e.g. cytokeratin8/18, p53, PAI-2 and in particular the urokinase receptor uPAR). Thishas previously been very difficult with the known methods especially inthe case of tissue samples such as bone marrow aspirates. A furthermajor advantage of the newly developed method is that it allows aquantitative determination of the number and intensity of fluorescentcells for example with a confocal laser scanning microscope.

[0013] Step (a) of the method according to the invention comprises theprovision of a biological sample to be tested. For this a sample istaken from the patient e.g. from a body fluid such as blood or from atissue such as bone marrow. The method according to the invention isparticularly preferably used to detect disseminated tumour cells ofepidermal origin in the bone marrow. The bone marrow can be taken fromthe iliac crest bone. Mononuclear cells including tumour cells are thenpreferably concentrated in the sample. This concentration can be carriedout by known methods for example by density gradient centrifugation e.g.Ficoll in which a separation of erythrocytes and granulocytes occurs.

[0014] The sample to be tested preferably contains at least 10⁶ cells inorder to enable a reliable detection of rare cells. The sampleparticularly preferably contains 10⁶ to 10⁹ and in particular 5×10⁶ to5×10⁷ cells.

[0015] According to step (b) the sample is contacted with at least twodifferent binding molecules that are directed against the cells to bedetected. The binding molecules are preferably antibodies or antibodyfragments and in particular monoclonal antibodies or antibody fragments.However, it is also possible to use ligands of receptors such as the uPAreceptor that specifically occur in the cells to be detected. Examplesof such ligands are linear or/and cyclic peptides or peptide mimeticswhich can carry a fluorescent label.

[0016] The sample is preferably contacted with the fluorescent-labelledbinding molecules after the cells have been fixed on a solid phase. Thisfixation can be carried out by known methods e.g. using formaldehyde orglutardialdehyde. A microscope slide can for example be used as thesolid phase.

[0017] If necessary the cells present in the sample to be tested can bepermeabilized using a detergent such as a saponin. This enables thebinding molecules to also bind to intracellular determinants.

[0018] For the detection of tumour cells the binding molecules aredirected against determinants which only occur in tumour cells or arepresent at an increased concentration in tumour cells in the sample tobe tested but do not occur in normal cells or only in a lowconcentration. A structure from the interior of the cells e.g. acytokeratin is preferably selected as the first determinant.Cytokeratins are specific components of the cytoskeleton of epithelialcells and are not expressed in mononuclear blood or bone marrow cellswhich are of mesenchymal origin. Hence the presence of cytokeratins incells which have been taken from blood and bone marrow indicates thepresence of epithelial tumour cells. Examples of suitableanti-cytokeratin antibodies are the antibody A45B/B3 (Micromet GmbH,Munich, Germany) or the antibody CK2 (Boehringer Mannheim GmbH,Mannheim, Germany). Other detection antibodies directed againstintracellular tumour-associated antigens are known and are commerciallyavailable from various companies.

[0019] A structure on the cell surface such as a membrane receptor ispreferably selected as the second determinant. The urokinase receptor(uPAR) is a particularly preferred tumour-specific determinant. Thisreceptor can for example be detected using anti-uPAR antibodies such asIID7 and IIIF10 (Luther et al., Am. J. Path. 150 (1997), 1231-1244).Those anti-uPAR antibodies are preferably selected which have anaffinity for a tumour cell-specific uPAR which is at least comparable tothat for a uPAR from normal cells. Examples of anti-uPAR antibodieswhich also bind to tumour cells with high affinity are antibodies whichrecognize the epitope 52-60 of uPAR such as the above-mentioned antibodyIIIF10.

[0020] In contrast other anti-uPAR antibodies often only poorlyrecognize uPAR on tumour cells.

[0021] On the other hand uPAR can also be detected withfluorescent-labelled receptor ligands e.g. urokinase, urokinasefragments or urokinase peptides. Such detection methods are describedfor example by Chucholowski et al. (Fibrinolysis 6, Suppl. 4 (1992),95-102), Ciccocioppo et al. (J. Histochem. Cytochem. 45 (1997),1307-1313) and Luther et al. (Am. J. Pat. 150 (1997), 1231-1242).

[0022] At least two different fluorescent labelling groups are used inthe method according to the invention. It is advantageous to usefluorescent labelling groups which have emission spectra (e.g.red/green) that can be distinguished from one another. Examples ofsuitable fluorescent dyes are fluorescein and derivatives thereof,phycoerythrin, rhodamine, TRITC-amines, Texas Red® amines, CY3 and CY5as well as Alexa® 488 and Alexa® 568 (Molecular Probes). The fluorescentdyes can be directly e.g. covalently conjugated with the primary bindingmolecules that are specific for the cells to be detected. This isreferred to as a direct label. On the other hand the fluorescent dyescan be conjugated to secondary binding molecules which are in turndirected against the primary binding molecules. This is referred to asan indirect label. Both labelling methods or combinations thereof can beused in the method according to the invention.

[0023] The various binding molecules can be sequentially or concurrentlyincubated with the cell. An incubation with several binding molecules inparallel (primary binding molecules and optionally secondary bindingmolecules in the case of an indirect label) leads to a considerable timesaving.

[0024] The sample is evaluated by determining the fluorescence afterexciting the fluorescent labelling groups. A confocal laser scanningmicroscope or a fluorescence microscope is particularly preferably usedfor this which enable an evaluation of the sample by concurrent or/andsequential determination of the various fluorescent labelling groups.

[0025] The double-fluorescence labelling technique according to theinvention additionally enables a characterization of the cellsidentified as positive by reaction with the binding molecules. Thischaracterization can comprise site-specific or/and quantitativeevaluation of the label. Hence individual cells can be “scanned” bydetermining the label in several e.g. 10 to 50 planes of sectionsthrough the cell at distances of for example 0.1 to 1 μm. In additionthe determinants in the cell that have reacted with the bindingmolecules can be determined quantitatively on the basis of a standardcurve which has been constructed by measuring microparticles of adefined size and a defined amount of fluorescent dye.

[0026] The method according to the invention allows valuable diagnosticdata to be obtained from tumour patients and hence enables a sensitiveprognosis to be made for patients after operation of a primary tumour.

[0027] Finally the invention concerns a reagent kit for the detection ofcells in a biological sample comprising

[0028] (a) a first binding molecule which recognizes the cells to bedetected and a first fluorescent labelling group,

[0029] (b) a second binding molecule which recognizes the cells to bedetected and a second fluorescent labelling group, the first and thesecond binding molecule and the first and the second fluorescentlabelling group being different and

[0030] (c) means for fixing cells on a solid phase.

[0031] It was surprisingly found that uPAR antibodies which are directedagainst the epitope 52-60 of uPAR recognize a uPAR having aglycostructure that occurs in tumour cells i.e. bind to a uPAR expressedby tumour cells with an at least comparable affinity to a uPAR expressedby normal cells. In contrast other anti-uPAR antibodies e.g. HD13.1(Todd et al., CD87 workshop panel report. In: Kishimoto T. et al., publ.Leucocyte Typing VI, New York & London, Garland Publishing, Inc. 1997;1016-1020) only have a low affinity for uPAR from tumour cells.

[0032] Hence the invention concerns the use of an antibody or of anantigen-binding fragment thereof (preferably of a monoclonal antibody orof an antigen-binding fragment thereof) which is directed against theepitope 52 to 60 of uPAR to produce a diagnostic or therapeutic agentdirected against uPAR on tumour cells. Such antibodies like the knownmonoclonal antibody IIIF10 (Luther et al. (1997), supra) or antibodieshaving an equivalent binding specificity such as chimerised or humanizedantibodies or corresponding recombinant or proteolytic antibodyfragments, e.g. single-chain antibody fragments, recognize a uPARexpressed by tumour cells with an adequate affinity for diagnostic andtherapeutic purposes.

[0033] Furthermore it was surprisingly found that such antibodies orfragments thereof can be used as a diagnostic agent to predict thecourse of malignant diseases especially in the case of tumours e.g.breast carcinomas. In tumour samples from over 200 examined femalebreast carcinoma patients it was found that the binding of the antibodyIIIF10 or of a corresponding antibody with an equivalent bindingcapability has a significant prognostic relevance for the course of thedisease i.e. absence of recidivity or death. In this connection highantigen values indicate a shorter absence of recidivity or an earlierdeath. Such a prognostic significance was not found with antibodieswhich are directed against other regions of uPAR.

[0034] Due to their high affinity for tumour uPAR these antibodies orfragments thereof are also suitable as diagnostic agents for detectingtumour cells in a biological sample and in particular for detectingdisseminated tumour cells in bone marrow. Such detection methods can forexample be carried out as an ELISA or as previously elucidated in detaildouble-fluorescence detection methods.

[0035] Moreover antibodies which are directed against the epitope 52 to60 of uPAR or fragments thereof are suitable for preparing a therapeuticagent with for example selective function blocking activity iN tumourcells. In addition the antibodies or fragments thereof can be used inthe form of conjugates with a cytotoxic group to inhibit the growth ofor kill tumour cells. Examples of suitable cytotoxic groups areradioactive groups, toxins and cell growth inhibitors. For therapeuticapplications it is preferable to use chimeric antibodies with humanizedconstant domains the production of which is described for example inEP-B-0 120 694.

[0036] Yet a further subject matter of the invention are recombinantnucleic acids which code for a polypeptide with antibody properties andcontain the CDR3-VH sequence or/and the CDR3-VL sequence of the antibodyIIIF10. The CDR3 region of the VH cDNA is shown in SEQ ID NO.1/2 fromnucleotide 295 to 321 (corresponding to amino acid 99 to 107). The CDR3region of the VL cDNA is shown in SEQ ID NO.3/4 from nucleotide 265 to291 (amino acid 89 to 97). In addition the nucleic acids preferablycontain the sections of VH or/and VL cDNA coding for the CDR1 and/orCDR2 regions. The sequences for the CDR1-VH region are shown in SEQ IDNO.1/2 from nucleotide 91 to 105 (corresponding to amino acid 31 to 35,i.e. SYDIN). In SEQ ID NO.3/4 the CDR1 region of the VL cDNA extendsfrom nucleotide 70 to 102 (corresponding to amino acid 24 to 34, i.e.KAS . . . TVA). The CDR2 region of the VH cDNA extends from nucleotide148 to 198 (amino acid 50 to 66, i.e. WIF . . . FKD) in SEQ ID NO.1/2.The CDR2 region of the VL cDNA extends from nucleotide 148 to 168(corresponding to amino acid 50 to 56, i.e. LASNRHT) in SEQ ID NO.3/4.

[0037] Thus the invention concerns in particular recombinant nucleicacids which code for a polypeptide having antibody properties comprising

[0038] (a) a CDR3-VH sequence coding for the amino acid sequence (I):

[0039] D G S M G G F D Y or/and

[0040] (b) a CDR3-VL sequence coding for the amino acid sequence (II):

[0041] L Q H W N Y P Y T

[0042] Furthermore the invention concerns recombinant polypeptideshaving antibody properties comprising

[0043] (a) a CDR3-VH amino acid sequence (I):

[0044] D G S M G G F D Y or/and

[0045] (b) a CDR3-VL amino acid sequence (II):

[0046] L Q H W N Y P Y T

[0047] The recombinant nucleic acids and polypeptides preferably containthe CDR3 regions of the VH as well as of the VL sequence. Therecombinant polypeptides are particularly preferably single-chainantibodies e.g. scFv antibody fragments. In the recombinant polypeptidesthe framework domains which are not directly responsible for antigenbinding are preferably replaced by corresponding human sequences suchthat humanized antibody fragments are formed. The recombinantpolypeptides according to the invention can be coupled with effectorgroups i.e. cytotoxic groups for therapeutic applications or/anddetection groups for a tumour imaging.

[0048] The invention is further elucidated by the following figures andexamples.

[0049]FIG. 1: shows a diagrammatic view of the scanning of a cell in alaser microscope.

[0050] a) A total of 30 serial sections with a spacing of 0.5 μm isprepared from a ca. 15 μm large tumour cell.

[0051] b) The fluorescence is measured in each plane of the section andthen all fluorescence values are added.

[0052] c) The total fluorescence is calculated from a standard curve(latex microparticles containing a defined amount of fluorochrome).

[0053]FIG. 2: shows the result of the fluorescence staining of a tumourcell with the anti-cytokeratin antibody A45 B/B3 and Alexa 488 as afluorescent dye.

[0054] a) The sequence of images shows 24 photographs of a scanprocedure in which a ca. 12 μm breast carcinoma cell (ZR75) was measuredin section planes with a spacing of 0.5 μm in each case.

[0055] b) shows an extended focus photograph in which the totalintensity of the entire scan (a) has been projected onto a single imageplane.

[0056]FIG. 3: shows the result of an indirect fluorescence staining withA45B/B3 as the primary antibody and a secondary antibody conjugated withAlexa 488 (enlargement ×63),

[0057] a) transmission image

[0058] b) a cytokeratin-positive cell in the bone marrow smear of afemale patient with breast carcinoma.

[0059]FIG. 4: shows the result of a direct fluorescence staining with aconjugate of the antibody A45B/B3 and the fluorescent dye Alexa 488(enlargement ×63),

[0060] a) transmission image

[0061] b) cytokeratin detection in a mixed preparation of MCF7 tumourcells and peripheral blood lymphocytes (1:20).

[0062]FIG. 5: shows the result of a direct fluorescence staining with aconjugate of the anti-uPAR antibody IIIF10 and the fluorescent dye Alexa568 (enlargement ×63),

[0063] a) transmission image

[0064] b) uPAR receptor detection in a mixed preparation of MCF7 tumourcells and peripheral blood lymphocytes (1:20).

[0065]FIG. 6: shows the result of a direct double-fluorescence stainingwith the conjugates A45B/B3-Alexa 488 (anti-cytokeratin) andIIIF10-Alexa 568 (anti-uPAR),

[0066] a) transmission image

[0067] b) cytokeratin detection

[0068] c) uPAR receptor detection

[0069]FIG. 7: shows the result of the measurement of a tumour cell inthe bone marrow (enlargement ×63),

[0070] a) transmission image (Nomarski optics)

[0071] b) reaction of the cell with a conjugate of Alexa 488 and-ananti-cytokeratin antibody.

[0072] c) reaction of the cell with a conjugate of Alexa 568 and a uPARantibody. The cell nucleus is not stained. The reaction of the anti-uPARantibody is mainly limited to the cell membrane. The uPAR-positive bonemarrow cell which is negative for cytokeratin is shown on the bottomright. All other cells are uPAR-negative.

[0073]FIG. 8: shows the influence of uPA on the uPAR determination

[0074] a) the UPA/uPAR ratio in tumour extracts from 599 breastcarcinoma patients,

[0075] b) the determination of uPAR in the presence of different amountsof uPA.

[0076]FIG. 9: shows the uPAR-antigen content in various cells determinedby different test procedures:

[0077] IIIF10/HU277 black, HD13.1/HU277: dark grey, ADI light grey

[0078] a) normal cells

[0079] b) well-differentiated tumour cells

[0080] c) poorly-differentiated tumour cells

[0081]FIG. 10: shows the prognostic relevance of the uPAR antigencontent determined by various test procedures in 203 breast carcinomapatients

[0082] a) IIIF10/HU277

[0083] b) HD13.1/HU277

[0084] c) ADI

[0085]FIG. 11: shows the dose-dependent inhibition of tumour growth ofhuman breast cancer in naked mice by administering the antibody IIIF10.

[0086]FIG. 12: shows the binding of scFv IIIF10 to immobilized antigens.

[0087]FIG. 13: shows the inhibition of the binding of IIIF10 (monoclonalantibody/moab and scFv) to uPAR by peptides.

[0088] SEQ ID NO 1/2: shows the nucleotide sequence of the cDNA codingfor the VH chain of IIIF10 VH and the corresponding amino acid sequence.

[0089] SEQ ID NO 3/4: shows the nucleotide sequence of the cDNA codingfor the VL chain of IIIF10 and the corresponding amino acid sequence.

EXAMPLES 1. Double-Fluorescence Determination of Tumour Cells

[0090] 1.1 Material

[0091] The monoclonal mouse antibody A45B/B3 (Kaspar et al., Eur. J.Cancer Clin. Oncol 23, (1987), 137-147) is directed against thecytokeratin filaments 8, 18 and 19 (CK 8, 18, 19). This antibody wasdirectly conjugated with the fluorochrome ALEXA 488 from MolecularProbes. The uPA receptor is specifically detected by the monoclonalmouse antibody IIIF10 (Luther et al. (1997), supra) (epitope 52 to 60).The monoclonal antibodies HD13.1 and IID7 (Luther et al. (1997), supra)(epitope 125 to 132) as well as the polyclonal rabbit antibody #399R(Stahl et al., Cancer Res. 54 (1994), 3066-3071) and the chickenantibody HU277 (Magdolen et al., Electrophoresis 16 (1995), 813-816) areavailable as additional uPA receptor antibodies. All monoclonalantibodies against the uPA receptor were directly conjugated with thefluorescent dye ALEXA® 568. TABLE 1 Directly conjugated antibodies thatwere used exci- tation directly range in Monoclonal conjugated the Manu-antibody Antigen with CLSM* facturer mAb II D 7 uPAR, ALEXA 568 568 nmPathology (mouse) domain 2 ( ™Molecular Dresden and Probes) Gynae-cological Hospital Munich mAb III F 10 uPAR, ALEXA 568 568 nm Pathology(mouse) domain 1 ( ™Molecular Dresden and Probes) Gynae- cologicalHospital Munich mAb HD 13.1 uPAR, ALEXA 568 568 nm Immunology (mouse)domains 2 + 3 ( ™Molecular Heidelberg Probes) mAB A45 cytokeratin ALEXA488 488 nm Micromet B/B3 8/9/18 ( ™Molecular Munich (mouse) Probes)

[0092] 1.2 Bone Marrow Preparations

[0093] A Jamshidi puncture is carried out in the operating theatre. 4-6ml bone marrow is taken from both iliac crests. The tumour cells in themononuclear cell fraction are concentrated by means of a Ficollgradient. 8 to 12 cytospins (10⁶ cells by cytospin) are prepared perpatient. After air-drying the preparations are fixed and permeabilized.

[0094] 1.3 Fixation and Permeabilization

[0095] 1. Fixation in 4% paraformaldehyde (PFA) for 30 min.

[0096] 2. Wash three times in phosphate-buffered saline/1% bovine serumalbumin (PBS/BSA).

[0097] 3. Permeabilize in 0.025% saponin for 45 min.

[0098] 4. Wash three times in PBS/1% BSA.

[0099] 1.4 Double-Labelling of the Cytokeratin and uPA Receptor

[0100] 1.4.1. Indirect Method

[0101] 1. Incubate overnight with the primary mouse antibody A45B/B3(final concentration 0.004 mg/ml) in PBS/1% BSA.

[0102] 2. Wash three times with PBS/1% BSA.

[0103] 3. Incubate for two hours with the second primary rabbit antibody#399 R (final concentration 0.05 mg/ml) diluted in PBS/1% BSA.

[0104] 4. Wash three times in PBS/1% BSA.

[0105] 5. Secondary antibody goat anti-mouse-Alexa 488 (finalconcentration 0.02 mg/ml) diluted in PBS/1% BSA, incubation period 30min.

[0106] 6. Wash three times in PBS/1% BSA.

[0107] 7. Secondary antibody goat anti-rabbit-Alexa 568 (finalconcentration 0.02 mg/ml) diluted in PBS/1% BSA, incubation period 30min.

[0108] 8. Wash three times in PBS/1% BSA.

[0109] 9. Cover with 5 μl PBS/1% BSA and examine under a microscope.

[0110] 1.4.2 Direct Method

[0111] 1. Incubate for 1 hour with the antibody A45B/B3-Alexa 488 (finalconcentration 0.0014 mg/ml) diluted in PBS/1% BSA.

[0112] 2. Wash three times in PBS/1% BSA.

[0113] 3. Incubate for 1 hour with the antibody IIIF10-Alexa 568 (finalconcentration 0.003 mg/ml) diluted in PBS/1% BSA.

[0114] 4. Wash three times in PBS/1% BSA.

[0115] 5. Cover with 5 μl PBS/1% BSA and examine under a microscope.

[0116] 1.5 Quantification

[0117] The antigens reacting with the fluorescent antibody arevisualized in a confocal laser scanning microscope at an excitationrange of 488 nm and 568 nm. The tumour cells are divided into 20 to 30planes of section by scanning the cell in a laser microscope i.e. bylayering in 0.5 μm steps. All fluorescences are detected and the sum ofthese measurements is calculated. The antigens in the tumour cell whichhave reacted with the antibody can be quantified on the basis of astandard curve which has been previously constructed by measuring latexbeads containing a defined amount of fluorescent dye.

[0118]FIG. 1 shows a diagram of the principle of the scanning procedureused to localize and quantify the fluorescent label. FIGS. 2 to 7 showexamples of results for the practical application of the methodaccording to the invention.

2. Tumour Specificity of the Monoclonal Antibody IIIF10

[0119] Two different ELISA systems were developed for the detection ofuPAR antigen:

[0120] 1) Capture antibody: polyclonal chicken antibody HU277 (Magdolenet al. (1995), supra); detection antibody: monoclonal antibody IIIF10(Luther et al. (1997), supra)

[0121] 2) Capture antibody: polyclonal chicken antibody HU277;monoclonal antibody HD13.1 (Todd et al. (1997), supra).

[0122] These ELISA systems were compared with a commercially availableELISA (ADI) for uPAR (American Diagnostica Inc. Greenwich, Conn. USA).

[0123] The tested ELISA systems were matched using recombinantaffinity-purified human uPAR (rec-uPAR) expressed in CHO cells. Allthree ELISA systems exhibited a comparable linearity and sensitivitytowards rec-uPAR.

[0124] In further experiments it was demonstrated that the actual uPARantigen content on cells can also be determined in the presence of an upto six-fold excess of uPAR. The recovery was >95% in the case ofIIIF10/HU277 and the HD13.1/HU277 test and >80% in the case of the ADItest. The uPA/uPAR ratio in 599 analysed tumour extracts is typically <3in 95% of the cases (tests with ADI-UPA and ADI-uPAR-ELISA). Theseresults are shown in FIG. 8.

[0125] Subsequently the uPAR antigen contents were determined in lysatesof various cell types. This showed that the determination of uPARantigen in non-malignant cells (e.g. keratinocytes [HaCaT], endothelialcells from the umbilical cord [HUVEC], epithelial cells from the breast[HMEC] gave comparable results in all three ELISA systems. In contrastthe situation was quite different in the case of tumour cell lines. Inwell-differentiated breast carcinoma cells only the IIIF10/HU277 ELISAdetected significant amounts of tumour-associated uPAR whereas inpoorly-differentiated breast carcinoma cell lines the IIIF10/HU277 andthe ADI-ELISA gave comparable values. The HD13.1/HU277-ELISA detectedtoo little uPAR in well-differentiated as well as inpoorly-differentiated carcinoma cells. The data are shown in FIG. 9.

3. Prognostic Relevance of the Monoclonal Antibody IIIF10

[0126] In a clinical study the uPAR antigen content was determined usingall three ELISA systems described in example 2 in tumour samples fromover 200 breast carcinoma patients. This showed that the antigen valuesmeasured with the IIIF10/HU277-ELISA have a significant prognosticrelevance for the course of the disease i.e. for the absence ofrecidivity or death. Such a prognostic relevance was not found with thetwo other ELISA systems. The data are shown in FIG. 10.

4. In vivo Effect of the Monoclonal Antibody IIIF10

[0127] 4 to 6 week old Balb/C/3 naked mice were injected on the rightflank with 6×10⁶ human breast cancer cells MDA-MB231 (Price et al.,Cancer Res. 50 (1990), 717-721) in a total volume of 300 μl. Beforeinjection the cancer cells were mixed in each case with 200 μg of themurine monoclonal antibody IIIF10 in PBS, pH 7.4. Subsequently the micewere treated intraperitoneally with the monoclonal antibody IIIF10 at adose of 2 mg/kg body weight or 10 mg/kg body weight in an injectionvolume of 300 μl. The volume of the primary tumours in cm³ occurring inthe mice was determined after four weeks by measuring the two largestdiameters of the tumours. PBS pH 7.4 was administered to the controlmice, each group consisted of six mice.

[0128] The results are shown in FIG. 11. It can be seen that theadministration of the antibody greatly reduced the growth of primarytumours. The inhibition of growth was even more pronounced when 10 mg/kgbody weight was administered than when a dose of 2 mg/kg body weight wasadministered.

5. Preparation of Recombinant Monoclonal Antibody IIIF10

[0129] mRNA from hybridoma cells producing IIIF10 was isolated andtranscribed into cDNA. The cDNA fragments coding for the variableregions of the heavy (VH) and the light (VL) chain were amplified byRT-PCR using gene-specific primers. The VH and VL gene segments werecloned into a phagemid vector to enable expression of the variableregions as a single-chain antibody (scFv). The scFv molecules werepresented by phage display on the surface of filamentous phages as afusion protein containing the small phage coat protein pIII. Phageswhich exhibited a functional expression of scFv-FIII10 were selected byspecific binding of uPAR. The selected phages were used to infect E.coli cells which enabled the production and secretion of soluble scFvmolecules into the culture medium. FIG. 12 shows the binding of the scFvsupernatant to uPAR immobilized on a solid phase. The binding capabilityof the antibodies scFv-anti-X and scFv-anti-Y was also tested forcontrol purposes.

[0130] In order to further test the binding specificity, peptides wereused which had been used to map the epitope of the antibody IIIF10(Luther et al., J. Pathol 150 (1997), 1231-1244). As can be seen in FIG.13 only one peptide the sequence of which contains the complete IIIF10epitope on uPAR (51-65), can prevent the binding of the monoclonalantibody and of scFvIIIF10 to uPAR. Another peptide with an incompletesequence epitope (48 to 59) is >100-fold less effective. None of thepeptides can prevent the binding of a control antibody scFv-anti-X toits target protein X.

[0131] The nucleotide sequence of VH cDNA and the corresponding aminoacid sequence are shown in SEQ ID NO. 1/2. The nucleotide sequence ofthe VL cDNA and the corresponding amino acid sequence are shown in SEQID No. 3/4.

1 6 1 354 DNA Artificial Sequence phage sequence 1 cag gtg caa ctg cagcag tca gga cct gag ttg gtg aag cct ggg gct 48 Gln Val Gln Leu Gln GlnSer Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 tta gtg aag ata tcctgc aag gct tct ggt tac agt ttc aca agc tac 96 Leu Val Lys Ile Ser CysLys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 gat ata aat tgg gtg aagcgg agg cct gga cag gga ctt gag tgg att 144 Asp Ile Asn Trp Val Lys ArgArg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga tgg att ttt cct gga gatggt agt acc aat tac aat gag aaa ttc 192 Gly Trp Ile Phe Pro Gly Asp GlySer Thr Asn Tyr Asn Glu Lys Phe 50 55 60 aag gac aag gcc aca ctg act gctgac aaa tcc tcc agc aca gcc tac 240 Lys Asp Lys Ala Thr Leu Thr Ala AspLys Ser Ser Ser Thr Ala Tyr 65 70 75 80 atg cag ctc aac agc ctg act tctgag aac tct gca gtc tat ttc tgt 288 Met Gln Leu Asn Ser Leu Thr Ser GluAsn Ser Ala Val Tyr Phe Cys 85 90 95 gca aga gat gga agt atg ggg ggg tttgac tac tgg ggc caa ggg acc 336 Ala Arg Asp Gly Ser Met Gly Gly Phe AspTyr Trp Gly Gln Gly Thr 100 105 110 acg gtc acc gtc tcc tca 354 Thr ValThr Val Ser Ser 115 2 118 PRT Artificial Sequence phage sequence 2 GlnVal Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30Asp Ile Asn Trp Val Lys Arg Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Phe Pro Gly Asp Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 7580 Met Gln Leu Asn Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys 85 9095 Ala Arg Asp Gly Ser Met Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100105 110 Thr Val Thr Val Ser Ser 115 3 324 DNA Artificial Sequence phagesequence 3 gat gtt ttg atg acc caa act cca aaa ttc atg tcc aca tca gtagga 48 Asp Val Leu Met Thr Gln Thr Pro Lys Phe Met Ser Thr Ser Val Gly 15 10 15 gac agg gtc agc atc acc tgc aag gcc agt cag aat gtt cgt act act96 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Thr 20 2530 gta gcc tgg tat caa gag aaa cca ggg cag tct cct aaa gca ctg att 144Val Ala Trp Tyr Gln Glu Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45tac ttg gca tcc aac cgg cac act gga gtc cct gat cgc ttc aca ggc 192 TyrLeu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 agtgga tct gga aca gat ttc act ctc acc att agc aat gtg caa tct 240 Ser GlySer Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 gaagac ctg gca gat tat ttc tgt ctg caa cat tgg aat tat ccg tac 288 Glu AspLeu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Tyr 85 90 95 acg ttcgga ggg ggc acc aag ctg gaa atc aaa cgg 324 Thr Phe Gly Gly Gly Thr LysLeu Glu Ile Lys Arg 100 105 4 108 PRT Artificial Sequence phage sequence4 Asp Val Leu Met Thr Gln Thr Pro Lys Phe Met Ser Thr Ser Val Gly 1 5 1015 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Thr 20 2530 Val Ala Trp Tyr Gln Glu Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 4045 Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 5560 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 7075 80 Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Tyr 8590 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 5 9 PRTArtificial Sequence CDR3-VH sequence 5 Asp Gly Ser Met Gly Gly Phe AspTyr 1 5 6 9 PRT Artificial Sequence CDR3-LH sequence 6 Leu Gln His TrpAsn Tyr Pro Tyr Thr 1 5

1. Method for detecting cells in a biological sample which comprises thefollowing steps: (a) preparing a sample to be tested, (b) contacting thesample with at least two different binding molecules which recognize thecells to be detected, the binding molecules being each labelled withdifferent fluorescent dyes and (c) determining the fluorescent labels inthe sample fixed on a solid phase.
 2. Method as claimed in claim 1,characterized in that tumour cells are detected.
 3. Method as claimed inclaim 1 or 2, characterized in that the detection is carried out in abone marrow sample.
 4. Method as claimed in one of the claims 1 to 3,characterized in that antibodies or antibody fragments or/and receptorligands are used as cell-specific binding molecules.
 5. Method asclaimed in one of the claims 2 to 4, characterized in that a firstcytokeratin-specific binding molecule and a second urokinasereceptor-specific binding molecule are used.
 6. Method as claimed in oneof the claims 1 to 5, characterized in that the binding molecules areindirectly labelled.
 7. Method as claimed in one of the claims 1 to 5,characterized in that the binding molecules are directly labelled. 8.Method as claimed in one of the claims 1 to 7, characterized in that thesample is evaluated by a confocal laser scanning microscope or by afluorescence microscope.
 9. Method as claimed in one of the claims 1 to8, characterized in that the sample is evaluated by parallel or/andsequential determination of the fluorescence of the various labelinggroups.
 10. Method as claimed in one of the claims 1 to 9, additionallycomprising a characterization of cells identified by reaction with thebinding molecules.
 11. Method as claimed in claim 10, characterized inthat the characterization comprises a site-specific or/and quantitativedetermination of the fluorescent label.
 12. Reagent kit for thedetection of cells in a biological sample comprising (a) a first bindingmolecule which recognizes the cells to be detected and a firstfluorescent labelling group, (b) a second binding molecule whichrecognizes the cells to be detected and a second fluorescent labellinggroup, the first and the second binding molecule and the first and thesecond fluorescent labelling group being different and (c) means forfixing cells on a solid phase.
 13. Use of the method as claimed in oneof the claims 1 to 11 or the reagent kit as claimed in claim 12 todetect micrometastases in biological samples.
 14. Use of an antibodywhich is directed against the epitope 52-60 of the urokinase receptor(uPAR) or of an antigen binding fragment thereof to produce a diagnosticor therapeutic agent directed against uPAR on tumour cells.
 15. Use asclaimed in claim 14 as a diagnostic agent to predict the course ofmalignant diseases.
 16. Use as claimed in claim 14 as a diagnostic agentto detect tumour cells in a biological sample.
 17. Use as claimed inclaim 16 to detect disseminated tumour cells in bone marrow.
 18. Use asclaimed in one of the claims 15 to 17 in an ELISA.
 19. Use as claimed inone of the claims 15 to 17 in a double-fluorescence detection method.20. Use as claimed in claim 14 as a therapeutic agent for blocking thefunction of tumour cells.
 21. Use as claimed in claim 14 in the form ofa conjugate with a cytotoxic group to inhibit the growth of or killtumour cells.
 22. Use as claimed in claim 21, characterized in that thecytotoxic group is selected from radioactive groups, toxins andinhibitors.
 23. Use as claimed in one of the claims 14 to 22,characterized in that the antibody is selected from the monoclonalantibody IIIF10, fragments thereof or antibodies or antibody fragmentshaving an equivalent binding specificity.
 24. Recombinant nucleic acidwhich codes for a polypeptide having antibody properties comprising (a)a CDR3-VH sequence coding for the amino acid sequence (I): D G S M G G FD Y or/and (b) a CDR3-VL sequence coding for the amino acid sequence(II): L Q H W N Y P Y T.
 25. Recombinant polypeptide having antibodyproperties comprising: (a) a CDR3-VH amino acid sequence (I): D G S M GG F D Y or/and (b) a CDR3-VL amino acid sequence (II): L Q H W N Y P YT.
 26. Recombinant polypeptide as claimed in claim 25, characterized inthat it is an scFv antibody fragment.
 27. Recombinant polypeptide asclaimed in claim 25 or 26, characterized in that it is a humanizedantibody fragment.
 28. Recombinant polypeptide as claimed in one of theclaims 25 to 27, characterized in that it is coupled to an effectorgroup.